Method for affinity viscosimetry and viscosimetric sensor

ABSTRACT

A method and apparatus for determining solute levels by affinity viscosimetry involving a sensitive fluid, in which the sensitive fluid flows continuously through a first hydraulic resistor in the flow direction of the dialysis chamber, and the sensitive fluid modified by dialysis simultaneously flows through another resistor, wherein the pressure differences between the resistors is determined on-line with the aid of pressure sensors and converted into a relative value which is approximately proportional to the concentration of solute.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application is a Continuation of U.S. patent applicationSer. No. 10/232,969, filed on Aug. 30, 2002, which claims priority toInternational Patent Application PCT/CH01/00126, filed on Feb. 28, 2001,which in turn claims priority to German Application No. DE 100 10 539A1, filed on Mar. 3, 2000, the contents of which are incorporated hereinby reference.

BACKGROUND

[0002] For measuring blood sugar concentration, viscosimetric affinitysensors have, among other things, been developed which can beminiaturized and used in an implanted form (DE 195 01 159) or astransdermal sensors (DE 197 14 087).

[0003] Viscosimetric affinity sensors for determining sugar levels arebased on a sensitive fluid, a concentrated solution consisting ofmacromolecular branched dextran and the tetravalent bonding proteinconcanavalin A (ConA), with the specificity of glucose, being situatedin a dialysis chamber coupled to a device for measuring viscosity. Theviscosity of the sensitive fluid is high when the dextran molecules arecross-linked via their exposed terminal glucose groups by ConA, and isreduced, dependent on concentration, with the free glucose penetratingthe dialysis chamber by diffusion from a glycosuric external solution.

[0004] A particularly favorable method of affinity viscosimetry involvesmeasuring the viscosity, once dialysis has been performed in the segmentof a microdialysis fiber, by measuring the flow resistor of a downstreamcapillary (DE 197 14 087). A known problem in affinity viscosimetry isthat the viscosity of the sensitive fluid is dependent not only on theconcentration of glucose but to a large extent also on the temperatureand on the concentration of the active glycopexic protein (Ballerstädtand Ehwald, Biosensors & Bioelectronics 9: 557-567, 1994; Ehwald et al.,Analytical Biochemistry 234: 1-8, 1996). In order to release the signalsof a viscosimetric affinity sensor for glucose from this significanttemperature-dependency, relative values having lowtemperature-dependency can be formed (Ballerstädt and Ehwald, Biosensors& Bioelectronics 9: 557-567, 1994). Up until now, only methods fordiscontinuously determining the relative values by consecutivelymeasuring the viscosity changed by glucose and the reference viscosityhave been known. A method for continuously determining such relativevalues in a sensor on-line has not been known up until now.

[0005] Developing a viscosimetric affinity sensor which operates on-linerequires a method for preparing readings which convertsviscosity-dependent measured values provided by the sensor on-line intoglucose concentration. In this connection, the aim is that the sensordetects a measured value which is directly dependent, in a linearrelationship, on the glucose concentration and is simultaneouslyindependent of the temperature and the concentration of active ConA inthe sensitive fluid. The method to this effect has not been known upuntil now.

SUMMARY

[0006] An object of the invention is to provide a method and a sensorfor determining sugar levels by affinity viscosimetry, which allow aparameter which is largely independent of the temperature and theconcentration of ConA and directly proportional to the concentration ofsugar to be detected on-line.

[0007] The object is addressed by the method for affinity viscosimetryand by a viscosimetric sensor in accordance with the present invention.

[0008] In one embodiment, the present invention comprises a method andapparatus for determining solute levels by affinity viscosimetryinvolving a sensitive fluid, in which the sensitive fluid flowscontinuously through a first hydraulic resistor in the flow direction ofthe dialysis chamber, and the sensitive fluid modified by dialysissimultaneously flows through another resistor, wherein the pressuredifferences between the resistors is determined on-line with the aid ofpressure sensors and converted into a relative value which isapproximately proportional to the concentration of solute.

[0009] In one embodiment, the present invention comprises a method andapparatus for determining sugar levels by affinity viscosimetry, inwhich the sensitive fluid flows continuously through a first hydraulicresistor in the flow direction of the dialysis chamber, and thesensitive fluid modified by dialysis simultaneously flows throughanother resistor, wherein the pressure differences between the resistorsis determined on-line with the aid of pressure sensors and convertedinto a relative value which is approximately proportional to theconcentration of sugar.

[0010] In accordance with the invention, it is advantageous if thesensitive fluid flows continuously, having a defined sugar content orhaving no sugar content, through a hydraulic resistor in the flowdirection of the dialysis chamber, the reference resistor, and thesensitive fluid modified by dialysis simultaneously flows throughanother resistor which is approximately isothermal with the referenceresistor, the measuring resistor, wherein the pressure difference whichdrops away across the reference resistor and the measuring resistor isdetermined on-line with the aid of pressure sensors and converted into arelative value approximately proportional to the concentration of sugar.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 depicts possible variants for the arrangements of theresistors in a sensor in accordance with the invention;

[0012]FIG. 2 is a schematic representation of a sensor in accordancewith the invention for determining sugar levels by affinityviscosimetry; and

[0013]FIGS. 3a and 3 b are diagrams showing relative viscosity andrelative fluidity against glucose concentration.

DETAILED DESCRIPTION

[0014]FIG. 1 schematically shows a few of the possible variants for thearrangements of the resistors, the dialysis chamber and the pressuresensors needed for measuring. The arrow represents the pressure pump(Variants 1, 2 and 4) or suction device (Variant 3) with the generatedflow direction of the sensitive fluid or solution.

[0015] The reference resistor Rr, the dialysis chamber D and themeasuring resistor Rm can lie in succession on a flow path (FIG. 1,Variants 1 to 3), or the reference resistor lies on one flow path andthe dialysis chamber and the measuring resistor lie together on aparallel flow path (FIG. 4, Variant 4).

[0016] If the reference resistor and the measuring resistor lie on oneflow path, one pump or suction device is sufficient, and the drop inpressure across the two resistors can be detected by a suitablearrangement of pressure sensors P1 and P2 (FIG. 1, Variants 1 to 3). Ifthe reference resistor and the measuring resistor lie on two parallelflow paths, they are connected in accordance with the invention to oneor more pump or suction devices which maintain a constant relationbetween the flows on the two flow paths (FIG. 1, Variant 4).

[0017] To simultaneously measure the drop in pressure across thereference resistor and the drop in pressure across the flow resistor,pressure sensors can be suitably arranged on the flow path, wherein thedeformable membranes of these pressure sensors must lie either betweenthe atmosphere and a measuring point on the flow path (FIG. 1, Variants1, 3 and 4) or between two different measuring points on the flow path(P1 in FIG. 1, Variant 2).

[0018] If the measuring resistor and the reference resistor are measuredsimultaneously, the ratio of measuring resistor and reference resistoris known to provide a relative, temperature-dependent viscosity(Ballerstädt and Ehwald, Biosensors & Bioelectronics 9: 557-567, 1994),which does not, however, decrease linearly with the glucoseconcentration and is not suitable for calculating glucose levels fromthe measured resistor values in a sensor. The relative fluidity (“RF”)calculated in accordance with the invention therefore represents thequotient between the drop in pressure at the reference resistor and thesum of the drop in pressure at the measuring resistor and a correctionvalue leading to a linearization of the correlation with the glucoseconcentration. The relative fluidity is a relative value which isindependent of temperature and ConA concentration and which has a linearrelationship to the glucose concentration.

[0019] It is important for forming the temperature-independent relativevalue for the flow resistors cited to be kept isothermal. This may beachieved by contacting the two resistors with the body or with anadditionally temperature-stabilized device, or by having the tworesistors in joint contact with a good heat conductor. For the method inaccordance with this embodiment, it is furthermore necessary for theexpandable volume content of the flow path between the referenceresistor and the measuring resistor to be smaller than the volume ofsensitive fluid moved by the pump or suction device within a particularperiod of time corresponding to the measuring task, since otherwise thechange in pressure at the measuring resistor responds too slowly to thechange in viscosity. This period of time should not be longer than 15minutes.

[0020] Exemplary Application:

[0021] In the equipment shown in FIG. 2, sensitive fluid is movedthrough a flow channel at a constant speed (5 μl/h) by means of a pump,wherein a pressure sensor for measuring the pressure p1, the hydraulicreference resistor 3, a pressure sensor 4 for measuring the pressure p2,a dialysis probe 5, the hydraulic measuring resistor 6 and a collectingvessel 7 for the used sensitive fluid are situated in succession on saidflow channel. In short intervals, the pressures p1 and p² are measuredsimultaneously and stored, assigned to the time of measurement, by meansof a programmable evaluation unit 8. The difference p1-p2 is then thedrop in pressure across the reference resistor, and p2 the drop inpressure across the measuring resistor. From these pressure values, theevaluation unit calculates the relative fluidity and/or, with the aid ofsettable constant calibration parameters, the glucose concentration, anddisplays this on a display.

[0022] If the quotient Q is the ratio of the drop in pressure across thereference resistor to the drop in pressure across the measuringresistor, RF may be calculated according to the formula:

RF=Q/(1+kQ)  (Equation 1)

[0023] where the constant k is a linearization parameter dependent onthe sensitive fluid and the ratio of the resistors, and is determinediteratively for the best correlation between the RF values and thecorresponding values of a glucose concentration series (FIG. 3b). Asopposed to the relative viscosity (FIG. 3a), the relative fluiditydefined by Equation 1 is proportional to the glucose concentration.

[0024] In the foregoing description embodiments of the invention havebeen presented for the purpose of illustration and description. They arenot intended to be exhaustive or to limit the invention to the preciseform or steps disclosed. Obvious modifications or variations arepossible in light of the above teachings. The embodiments were chosenand described to provide the best illustration of the principals of theinvention and its practical application, and to enable one of ordinaryskill in the art to utilize the invention in various embodiments andwith various modifications as are suited to the particular usecontemplated. All such modifications and variations are within the scopeof the invention as determined by the appended claims when interpretedin accordance with the breadth they are fairly, legally, and equitablyentitled.

1. A method for determining sugar levels by affinity viscosimetryinvolving a sensitive fluid, in which the sensitive fluid flowscontinuously through a first hydraulic resistor in the flow direction ofthe dialysis chamber, and the sensitive fluid modified by dialysissimultaneously flows through another hydraulic resistor, wherein thepressure differences between the resistors is determined on-line withthe aid of pressure sensors and converted into a relative value which isapproximately proportional to the concentration of sugar.
 2. The methodas set forth in claim 1, wherein said fluid exhibits one of a definedsugar content and no sugar content.
 3. The method as set forth in claim1, wherein said first resistor is a reference resistor and said anotherresistor is a measuring resistor which is approximately isothermal withsaid reference resistor, wherein the pressure difference which dropsaway across said measuring resistor and said reference resistor isdetermined.
 4. The method as set forth in claim 3, wherein saidreference resistor, said dialysis chamber and said measuring resistorare in succession on a flow path.
 5. The method as set forth in claim 3,wherein said reference resistor is on one flow path and said dialysischamber and said measuring resistor are together on a parallel flowpath.
 6. The method as set forth in claim 3, wherein the relative valuerepresents the quotient between the drop in pressure at said referenceresistor and the sum of the drop in pressure at said measuring resistorand a correction value leading to a linearization of the correlationwith the glucose concentration.
 7. A method for determining solutelevels by affinity viscosimetry involving a sensitive fluid, in whichthe sensitive fluid flows continuously through a first resistor in theflow direction of the dialysis chamber, and the sensitive fluid modifiedby dialysis simultaneously flows through another resistor, wherein thepressure differences between the resistors is determined on-line withthe aid of pressure sensors and converted into a relative value which isapproximately proportional to the concentration of solute.
 8. The methodas set forth in claim 7, wherein said first resistor is a referenceresistor and said another resistor is a measuring resistor which isapproximately isothermal with said reference resistor, wherein thepressure difference which drops away across said measuring resistor andsaid reference resistor is determined.
 9. The method as set forth inclaim 8, wherein the relative value represents the quotient between thedrop in pressure at said reference resistor and the sum of the drop inpressure at said measuring resistor and a correction value leading to alinearization of the correlation with the solute concentration.
 10. Adevice for determining solute concentration by affinity viscosimetryinvolving a sensitive fluid, comprising a first resistor in the flowdirection of a dialysis chamber which the sensitive fluid continuouslyflows through, another resistor which the sensitive fluid modified bydialysis simultaneously flows through, at least one pressure sensorwhich determines the pressure difference between said resistors on-line,and means for converting the pressure difference to a relative valueapproximately proportional to the concentration of solute.
 11. A sensorfor determining sugar levels by affinity viscosimetry involving asensitive fluid, comprising a first hydraulic resistor in the flowdirection of a dialysis chamber which the sensitive fluid continuouslyflows through, another hydraulic resistor which the sensitive fluidmodified by dialysis simultaneously flows through, pressure sensorswhich determine the pressure differences between said resistors on-line,and a device by means of which the pressure differences are convertedinto a relative value which is approximately proportional to theconcentration of sugar.
 12. The sensor as set forth in claim 11, furthercomprising one of a continuously conveying pump or suction device whichis coupled to a flow path with at least two pressure sensors, on whichflow path said first hydraulic resistor, the dialysis chamber and theanother hydraulic resistor are arranged in succession in the flowdirection, wherein the flow resistors are in joint contact with one of aperson's body, a temperature-stabilized device or a heat conductor, andthe expandable volume content of the flow path between said firsthydraulic resistor and said another hydraulic resistor is smaller thanthe volume of sensitive fluid moved by the pump or suction device within15 minutes.
 13. The sensor as set forth in claim 11, further comprisingtwo parallel flow paths coupled to one or more pump or suction deviceswhich maintain a constant relation between the flows on the two flowpaths; said first hydraulic resistor on one flow path and said dialysischamber and said another hydraulic resistor on the other flow path, saidanother hydraulic resistor downstream of the dialysis chamber; apressure sensor arranged on each of the two flow paths for measuring thedrop in pressure across the respective flow resistors, wherein theresistors are in joint contact with one of a person's body, atemperature-stabilized device or a heat conductor.
 14. The sensor as setforth in claim 12, wherein a deformable membrane of the pressure sensorslies between the atmosphere and a suitable measuring point on the flowpath, so measuring the drop in pressure across said resistors.
 15. Thesensor as set forth in claim 13, wherein a deformable membrane of thepressure sensors lies between suitable points on the flow paths, someasuring the drop in pressure across said resistors.
 16. A device fordetermining sugar levels by affinity viscosimetry involving a sensitivefluid, comprising a first resistor through which the sensitive fluidflows, a dialysis chamber through which the sensitive fluid flows,another resistor through which the sensitive fluid modified by dialysissimultaneously flows, at least one pressure sensor which determines thepressure difference between said resistors on-line, and means forconverting the pressure difference to a relative value approximatelyproportional to the sugar level.
 17. The device as set forth in claim16, further comprising one of a continuously conveying pump or suctiondevice which is coupled to the flow path of the sensitive fluid, onwhich flow path first resistor, the dialysis chamber and the anotherresistor are arranged in succession in the flow direction.
 18. Thedevice according to claim 17, wherein the resistors are in contact withone of a person's body, a temperature-stabilized device or a heatconductor.
 19. The device according to claim 16, further comprising twoparallel flow paths coupled to one or more pump or suction devices, saidfirst resistor on one flow path and said dialysis chamber and anotherresistor on the other flow path, said another resistor downstream ofsaid dialysis chamber, and a pressure sensor associated with each of theflow paths for measuring the drop in pressure across the respectiveresistors.
 20. The device according to claim 19, wherein the resistorsare in contact with one of a person's body, a temperature-stabilizeddevice or a heat conductor.